All mammals possess a hematopoietic (bloodforming) system that replenishes the multiplicity of blood cell types found in a healthy animal, including white blood cells (neutrophils, macrophages, and basophil/mast cells), clot forming cells (megakaryocytes, platelets), and red blood cells (erythrocytes). The average human male's hematopoietic system has been estimated to produce on the order of 4.5 .times.10.sup.11 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight [Dexter et al., (1985) BioEssays, vol. 2: 154-158]. Current scientific understanding proposes that small amounts of specific hematopoietic growth factors direct the proliferation, differentiation, and maturation of each of the various hematopoietic cell types from a small population of pluripotent hematopoietic stem cells. These various growth factors act at different times on different cell populations, ultimately giving rise to a functional hematopoietic system.
One specific and vital role of the mammalian hematopoietic system is the production of erythrocytes, or red blood cells, which transport oxygen to the various tissues of the animal's body. The process of producing erythrocytes ("erythropoiesis") occurs continuously throughout an animal's life span to offset erythrocyte destruction. The typical red blood cell has a relatively short life-span, usually 100 to 120 days; Gray's Anatomy, Williams et al. eds., Churchill Livingstone, 1989, p. 665. Erythropoiesis is a precisely controlled physiological mechanism whereby sufficient numbers of erythrocytes are produced to enable proper tissue oxygenation, but not so many as to impede circulation.
Erythropoiesis is now known to be primarily controlled by the polypeptide erythropoietin (EPO), an acidic glycoprotein. Erythropoietin is produced as the result of the expression of a single copy gene located in a chromosome of a mammal. A DNA molecule encoding a DNA sequence for human EPO has been isolated and is described in U.S. Pat. No. 4,703,008, hereby incorporated by reference, and hereinafter referred to as the "Lin patent." Also, DNA molecules coding for EPO from monkeys [Lin et al., (1986) Gene, vol. 44, pp: 201 -209] and mice [McDonald et al., (1986) Mol. Cell Biol., pp: 842] have been described. The amino acid sequence for recombinant human EPO ("rHuEPO") is identical to the sequence for EPO obtained from human urinary sources. However, as could be expected, the glycosylation of rHuEPO differs from that of urinary EPO and human serum EPO. See, e.g. Starring et al. (1992), J. Endocrin., vol. 134, pp: 459-484; Strickland et al. (1992), J. Cell. Biochem., suppl. 16D, p. 324; Wide et al. (1990), Br. J. Haematol., vol. 76, 121-127.
In a healthy mammal, EPO is present in the blood plasma in very low concentrations, as the tissues are being sufficiently oxygenated by the existing number of circulating erythrocytes. The EPO present stimulates the production of new erythrocytes to replace those lost to the aging process. Additionally, EPO production is stimulated under conditions of hypoxia, wherein the oxygen supply to the body's tissues is reduced below normal physiological levels despite adequate perfusion of the tissue by blood. Hypoxia may be caused by hemorrhaging, radiation-induced erythrocyte destruction, various anemias, high altitude, or long periods of unconsciousness. In contrast, should the number of red blood cells in circulation exceed what is needed for normal tissue oxygenation, EPO production is reduced.
However, certain disease states involve abnormal erythropoiesis. Until the advent of recombinant DNA technology, no EPO was available for therapeutic use. Today, the situation is different. Recombinant human EPO (rHuEPO) is being used therapeutically in a number of countries. In the United States, the U.S. Food and Drug Administration (FDA) has approved rHuEPO's use in treating anemia associated with end-stage renal disease. Patients undergoing hemodialysis to treat this disorder typically suffer severe anemia, caused by the rupture and premature death of erythrocytes as a result of the dialysis treatment. EPO is also useful in the treatment of other types of anemia. For instance, chemotherapy-induced anemia, anemia associated with myelodysplasia, those associated with various congenital disorders, AIDS-related anemia, and prematurity-associated anemia, may be treated with EPO. Additionally, EPO may play a role in other areas, such as helping to more quickly restore a normal hematocrit in bone marrow transplantation patients, in patients preparing for autologous blood transfusions, and in patients suffering from iron overload disorders. See e.g.U.S. Pat. No. 5,013,718, hereby incorporated by reference.
The effective use of EPO as a therapeutic agent requires that patients be administered small but highly precise doses of the protein in stable, pharmaceutically acceptable formulations. For an example of a current EPO formulation, see Sobata, J., Erythropoietin in Clinical Applications, Garnick, M., ed., Marcel Dekker, Inc., N.Y. (1990). Current therapy for end-stage renal disease calls for intravenous EPO administration within twelve hours of dialysis, three times a week. Alternatively, EPO may be administered to such patients by intravenous, intramuscular, intracutaneous, or subcutaneous injection.
The instant invention is based upon the unexpected discovery that EPO may be delivered in a therapeutically efficacious manner by direct administration of the protein to the lungs of a patient (hereinafter "pulmonary administration"). EPO delivered to the lung in this manner is absorbed into the patient's bloodstream for systemic distribution. This new route of EPO administration enables the rapid delivery of a specified medicament dosage to a patient without the necessity for injection. In addition, pulmonary administration more readily lends itself to self-administration by the patient.
There has been some prior success in the pulmonary administration of pharmaceutical compositions comprised of low molecular weight drugs, most notably in the area of beta-androgenic antagonists to treat asthma. Other low molecular weight, non-proteinaceous compounds, including corticosteroids and cromolyn sodium, have been administered systemically via pulmonary absorption. However, not all low molecular weight drugs can be efficaciously administered through the lung. For instance, pulmonary administration of aminoglycoside antibiotics, anti-viral drugs and anti-cancer drugs for systemic action has met with mixed success. In some cases, lack of delivery to the blood stream was attributed to the drug's inability to pass through the alveolar epithelium. In other cases, the drug was found to be irritating and bronchoconstrictive. Thus, even with low molecular weight substances, it is not at all predictable that the pulmonary delivery of such compounds will be an effective means of administration. See generally Peptide and Protein Drug Delivery, ed. V. Lee, Marcel Dekker, N.Y., 1990, pp. 1-11.
With respect to higher molecular weight pharmaceuticals, such as proteins, pulmonary delivery of such molecules is not unknown, although only a few examples have been quantitatively substantiated. Leuprolide acetate is a nonapeptide with luteinizing hormone releasing hormone (LHRH) agohist activity having low oral availability. Studies with animals indicate that inhalation of an aerosol formulation of leuprolide acetate results in meaningful levels in the blood. See Adjel et al., Pharmaceutical Research, Vol. 7, No. 6, pp. 565-569 (1990).
In contrast, Endothelin-1 (ET-1), a 21 amino acid vasoconstrictor peptide produced by endothelial cells, has been found to increase pulmonary inflation pressure but to have no significant effect on arterial blood pressure when administered by aerosol to guinea pigs. However, when administered intravenously, an important and sustained increase in arterial blood pressure was observed. See Braquet et al., Journal of Cardiovascular Pharmacology, Vol. 13, suppl. 5, s. 143-146 (1989).
The feasibility of delivering human plasma .alpha.1-antitrypsin to the pulmonary system using aerosol administration, with some of the drug gaining access to the systemic circulation, is reported by Hubbard et al., Annals of Internal Medicine, Vol. III, No. 3, pp. 206-212(1989).
Pulmonary administration of .alpha.-1-proteinase inhibitor to dogs and sheep has been found to result in passage of some of that substance into the bloodstream. See Smith et al., J. Clin. Invest., vol. 84, pp. 1145-1154 (1989). Likewise, aerosolized .alpha.-1 anti-trypsin diffused across the lung epithelium and entered into systemic circulation in sheep and humans. See Hubbard et al., (1989) Ann. Intern. Med., vol. 111, pp. 206-212. However, vasoactive intestinal peptide, a small polypeptide with a molecular weight of 3,450 daltons (D) which causes bronchodilation when given intravenously in animals, including humans, lacks efficacy when administered by inhalation. See Barrowcliffe et al., Thorax, vol. 41/2, pp. 88-93 (1986).
Experiments with test animals have shown that recombinant human growth hormone, when delivered by aerosol, is rapidly absorbed from the lung and produces faster growth comparable to that seen with subcutaneous injection. See Oswein et al., "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, 1990. Recombinant versions of the cytokines gamma interferon (IFN-.gamma.) and tumor necrosis factor alpha (TNF-.alpha.) have also been observed in the bloodstream after aerosol administration to the lung. See Debs et al., The Journal of Immunology, Vol. 140, pp. 3482-3488 (1988). Likewise, Pitt et al. have recently demonstrated the feasibility of pulmonary delivery of granulocyte-colony stimulating factor (G-CSF) to mammals. See commonlyowned U.S. patent application Ser. No. 07/669,792, filed Mar. 15, 1991.
Despite these examples of protein delivery via the pulmonary route, it is not predictable whether a particular polypeptide can be delivered for systemic effect in such a manner. Various factors intrinsic to the polypeptide itself, the delivery device, and particularly the lung, or a combination of these factors, can influence the success of pulmonary administration.
With respect to the lung, its complexity presents several barriers to pulmonary administration. Initially, after passing through the nose or mouth, inhaled air (and any particles contained therein) moves into the respiratory tree, which is composed of numerous dichotomous branches between the trachea and the alveoli. Bronchi, bronchioles, and terminal bronchioles comprise the conducting zone. The epithelium of these conducting airways is pseudo stratified and largely ciliated. The more distal levels of branching form the transitional and respiratory zones, comprised of respiratory bronchioles, alveolar ducts, and alveoli, where gas exchange and pulmonary absorbtion occur. The respiratory zone, in contrast to the conducting zone, is non-ciliated and comprised of a single cell layer.
In the normal human male, it is estimated that the lungs contain 3.times.10.sup.8 alveoli with a total surface area of approximately 140 m.sup.2. Alveoli are thin walled pouches that represent as minimal a barrier to gaseous exchange between the atmosphere and blood as is possible without comprising the integrity of the lung. Correspondingly, capillary beds adjacent to the alveoli are estimated to share a surface area of 125 m.sup.2 with the alveoli [Gray's Anatomy, supra]. Thus, each alveolus is in intimate association with numerous blood-bearing capillaries bringing oxygen-depleted blood from distal body tissues.
This air-blood barrier is comprised of the alveolar epithelium, the capillary endothelium, and the lymph-filled interstitial space separating these two cell layers. The mean thickness of the air-blood barrier in humans is 2.2 .mu.m [Gehr et al. (1978), Resp. Physiol., vol. 32, pp. 121-140], while the alveolar epithelium itself can be as thin as 0.05 .mu.m. In the alveolar epithelium, adjacent cells overlap and are bound by non-leaky tight junctions, which, in conjunction with the non-leaky single cell layer comprising the capillary endothelium, limits the movement of fluids, cells, salts, proteins, and numerous other macromolecules from the blood and intercellular spaces into the lumen of the alveoli. Most molecules, including proteins and polypeptides, must be actively or passively transported across this barrier in the absence of lung injury.
In addition, within the lung certain epithelial cells secrete mucous to form a contiguous aqueous lining throughout the lung to promote the diffusion of oxygen into the blood. This layer of moisture, with its incumbent surface tension within the alveoli, requires that a surfactant be secreted to reduce this surface tension. Otherwise, the alveoli would collapse. In mammals, this surfactant, comprised mostly of lipid, appears to be made up of five layers [Stratton, C. J., Cell Tissue Res., vol. 193, pp. 219-229 (1978)] and it can be a potential inhibitor of protein transport across the air-blood barrier.
Another protective system employed by the lung is the ciliary rejection current of the conducting zone. Here, numerous ciliated epithelial cells beat in a rhythmic one-way motion to propel the mucous lining overlaying the conducting airways towards the esophagus, where it is expelled from the respiratory system and moved into the digestive tract. Thus, particles impacting on these surfaces can be effectively removed prior to their penetration further into the lung.
Other cell types present in the alveolar lumen and in the interstitial space separating the alveolar epithelium from the capillary endothelium also serve as effective mechanisms for protecting the lung from and removing foreign material, such as protein-containing particles. Alveolar macrophages migrate from the blood across the air-blood barrier. These macrophages can phagocytose inhaled particles that reach the alveoli. These phagocytes may then migrate back into the lymphatic channels or to the base of the bronchial tree to be swept out of the lung by the ciliary rejection current. Additionally, other cell types, such as neutrophils and lymphocytes, can move into the alveoli from the blood to combat infection.
In view of the above, whether a particular therapeutic protein intended to have a systemic effect can be successfully delivered via the pulmonary route can not be predicted.